In order to continue to live, organisms have the ability to rapidly replace and repair lost or damaged cells and tissue, and this ability is known as “regenerative capacity”. Examples of “regenerative capacity” in higher animals include the commonly known phenomena of wound healing of skin and blood vessels, but even parenchymal organs such as the liver and kidneys are known to undergo cell growth and tissue reconstruction for rapid restoration of tissue homeostasis in response to tissue damage. Recent years have seen attempts to utilize this innate “regenerative capacity” of biological organisms to achieve cures or amelioration of various diseases and wounds, and such new medical techniques are coming to be known as “regenerative medicine”.
Stem cells play a central role in practicing “regenerative medicine”. “Stem cells” can be generally defined as undifferentiated cells having the ability to differentiate into specialized cells or polyfunctional cells, as well as having the ability to self-replicate, allowing repeated generation of cells identical to themselves. Unique stem cells are found in each tissue and cell type, and for example, blood cells such as erythrocytes, lymphocytes and megakaryocytes are produced via progenitor cells derived from stem cells known as “hematopoietic stem cells”, while skeletal muscle cells are produced from stem cells/precursor cells known as “satellite cells” and “myoblasts”. Additional types that have been identified to date include neural stem cells that are found in neural tissue such as the brain and spinal cord and produce neurons and glial cells, epidermal stem cells that produce epidermal cells and hair follicle cells, oval cells (hepatic stem cells) that produce hepatocytes and bile duct cells, and cardiac stem cells that produce cardiomyocytes.
Some regenerative medicine treatments using stem cells or precursor cells derived from such cells have already been implemented, and infusion graft methods with hematopoietic stem cells or hematopoietic precursor cells are well known for treatment of conditions caused by a lack or functional deficiency of blood cells, such as leukemia and a plastic anemia. However, stem cells present in parenchymal organs such as the brain, heart or liver are technically difficult to obtain from living tissues and/or to culture in vitro, and such stem cells also generally have low proliferation potency. Stem cells can also be recovered from tissues from corpses, but the medical use of cells obtained in this manner is associated with ethical problems. Consequently, regenerative treatments for neuropathy, cardiopathy and the like will require the development of techniques for generating desired cell types using cells other than stem cells present in such target tissues.
First, methods of utilizing “pluripotent stem cells” may be mentioned as strategies based on this approach. “Pluripotent stem cells” are defined as cells capable of prolonged or virtually indefinite proliferation in vitro while retaining their undifferentiated state, exhibiting normal karyotype (chromosomes) and having the capacity to differentiate into all cell types of the three germ layers (ectoderm, mesoderm and endoderm) under the appropriate conditions. Currently the most commonly known pluripotent stem cells are embryonic stem cells (ES cells) isolated from the early embryo, and the analogous embryonic germ cells (EG cells) isolated from fetal primordial germ cells, both of which are the subjects of ongoing research.
ES cells can be isolated as an undifferentiated stem cell population by transferring the inner cell mass of a blastocyst-stage embryo to in vitro culture and repeating the process of detaching and passaging the cell mass. The cells have suitable cell density on feeder cells prepared from primary cultured murine embryonic fibroblasts (hereinafter, MEF cells) derived from murine fetal tissue or stromal cells such as STO cells, and repeated passaging with frequent replacement of the culture medium can lead to establishment of a cell line retaining the property of undifferentiated stem cells. Another feature of ES cells is the presence of the enzyme telomerase, which exhibits an activity of maintaining chromosomal telomere length, and this enzyme confers to ES cells the capacity for virtually unlimited cell division in vitro.
ES cell lines produced in this manner are “pluripotent” as they can be repeatedly grown and passaged almost indefinitely while maintaining normal karyotype, and they are capable of differentiating into various different cell types. For example, when ES cells are transplanted into an animal body subcutaneously, intraabdominally or intratesticularly they form tumors called “teratomas”, but the tumors comprise a mixture of different cells and tissues including neurons, osteocytes, chondrocytes, intestinal cells, muscle cells and the like. In mice, intrauterine transplantation into a pseudopregnant mouse of an aggregate embryo generated by infusion graft of ES cells into a blastocyst-stage embryo or aggregation with an eight-cell stage embryo, results in generation of a “chimeric mouse”, which is an offspring possessing differentiated cells derived from the ES cells throughout the entire body or in parts of its organs and tissues. This technique is often used as a main method for generating “knockout mice” having certain genes which are artificially disrupted or modified.
It is also well known that ES cells are induced to differentiate into diverse types of cells by in vitro culturing as well. While the specific method differs depending on the type of cell, it is common to employ a method of inducing differentiation by forming an “embryoid body” (hereinafter, “EB”) which is a cell mass in an embryo-like state produced by aggregating ES cells by suspension culture. Such a method can produce cells having fetal stage endoderm, ectoderm and mesoderm characteristics, as well as differentiated cells such as blood cells, vascular endothelial cells, chondrocytes, skeletal muscle cells, smooth muscle cells, cardiomyocytes, glial cells, neurons, epithelial cells, melanocytes, keratinocytes, adipocytes and the like. Differentiated cells produced by in vitro culturing in this fashion have essentially the same structural and functional features as cells present in organs and tissues, and transplant experiments using experimental animals have demonstrated that ES cell-derived cells anchor to organs and tissues and function normally.
For reviews of ES cell properties and culturing methods, and their in vivo and in vitro differentiating abilities, refer to the following literature: Guide to Techniques in Mouse Development (Wasserman et al., Academic Press, 1993); Embryonic Stem Cell Differentiation in vitro (M. V. Wiles, Meth. Enzymol. 225:900, 1993); Manipulating the Mouse Embryo: A Laboratory Manual (Hogan et al., Cold Spring Harbor Laboratory Press, 1994)(Non-patent document 1); Embryonic Stem Cells (Turksen, ed., Humana Press, 2002)(Non-patent document 2).
EG cells can be produced by stimulating fetal germ cells known as primordial germ cells on feeder cells such as MEF cells or STO cells in the same manner as ES cells, using Leukemia Inhibitory Factor (hereinafter, LIF) and basic Fibroblast Growth Factor (hereinafter, bFGF/FGF-2), or chemical agents such as forskolin (Matsui et al., Cell 70:841, 1992; Koshimizu et al., Development 122:1235, 1996). It has been confirmed that EG cells have properties very similar to ES cells and have pluripotency (Thomson & Odorico, Trends Biotechnol. 18:53, 2000). Throughout the present specification, therefore, the term “ES cells” may include “EG cells”.
After Thomson et al. first established ES cells from a primate (rhesus monkey) in 1995, the concept of regenerative medicine using pluripotent stem cells began to approach the realm of possibility (U.S. Pat. No. 5,843,780; Proc. Natl. Acad. Sci. USA 92:7844, 1995). Later, the researchers used similar methods to successfully isolate and establish ES cell lines from human early embryos (Science 282:114, 1998). Research groups in Australia and Singapore later submitted similar reports (Reubinoff et al., Nat. Biotech. 18:399, 2000; International Patent Publication No. WO00/27995), and currently 20 different human ES cell lines have been registered at the U.S. National Institutes of Health (NIH)(http://stemcells.nih.gov/registry/index). Also, Gearhart and their colleagues have succeeded in establishing a human EG cell line from human primordial germ cells (Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998; U.S. Pat. No. 6,090,622).
When these pluripotent stem cells are used to produce research materials or regenerative medicine products, it is essential that the passaging methods used maintain the undifferentiated state and high proliferation potency of the cells. MEF cells or similar cells (such as STO cells) are usually used as feeder cells for ES/EG cells to maintain the undifferentiated state and high proliferation potency of the cells. Addition of fetal bovine serum (hereinafter, FBS) to the culture medium is also important, and it is crucial to select an FBS product which is suited for the culturing of the ES/EG cells, usually with the addition of FBS at about 10-20%. Also, LIF has been identified as a factor that maintains the undifferentiated state of ES/EG cells derived from mouse embryo (Smith & Hooper, Dev. Biol. 121:1, 1987; Smith et al., Nature 336:688, 1988; Rathjen et al., Genes Dev. 4:2308, 1990), and addition of LIF to culture can more effectively maintain the undifferentiated state (see the following literature: Manipulating the Mouse Embryo: A Laboratory Manual (Hogan et al., Cold Spring Harbor Laboratory Press, 1994 (Non-patent document 1) and Embryonic Stem Cells (Turksen ed., Humana Press, 2002)(Non-patent document 2)).
However, the culturing methods employed for these classical ES/EG cells are not suitable methods when human ES (or EG) cells are used for regenerative medicine or other practical purposes. One reason for this is that human ES cells are unresponsive to LIF, and lack of feeder cells causes death of the cells or loss of the undifferentiated state and differentiation into different cell types (Thomson et al., Science 282:1145, 1998). The use of feeder cells itself is another problem because as such co-culturing systems increase production cost and are poorly suited for large-scale culturing, while separation and purification of the ES cells from the feeder cells is required when the ES cells are to be actually used. In the future, when human ES cells and other pluripotent stem cells are utilized as cell sources for regenerative medicine, and particularly for cell transplantation therapy, the use of non-human animal cell products such as MEF cells and FBS will not be desirable because of risks including potential infection of the ES cells by heterozoic viruses and contamination with antigenic molecules that may be recognized as heteroantigens (Martin et al., Nature Med. 11:228, 2005).
Consequently, in order to refine ES/EG cell culturing methods and modify them to be suitable for future implementation, active efforts are being made to develop FBS substitutes (International Patent Publication No. WO98/30679) and to utilize human cells as feeders instead of MEF cells (Richards et al., Nature Biotech. 20:933, 2002; Cheng et al., Stem Cells 21:131, 2003; Hovatta et al., Human Reprod. 18:1404, 2003; Amit et al., Biol. Reprod. 68:2150, 2003).
Development of culturing methods using no feeders is another alluring prospect. Carpenter and coworkers have reported that seeding of ES cells in a Matrigel- or Laminin-coated culturing plate and addition of MEF cell conditioned medium to the culture medium allows prolonged culturing of human ES cells which retain their undifferentiated and pluripotency (Xu et al., Nature Biotech. 19:971, 2001 (Non-patent document 3); International Patent Publication No. WO01/51616 (Patent document 1)). The same group also succeeded in constructing a more effective ES cell culturing system by developing a serum-free medium containing added bFGF/FGF-2 or Stem Cell Factor (hereinafter, SCF)(International Patent Publication No. WO03/020920 (Patent document 2)). An ES cell culturing system using the same serum-free medium and requiring no feeder has also been reported by an Israeli research group (Amit et al., Biol. Reprod. 70:837, 2004 (Non-patent document 4)).
Recently, a method of maintaining the undifferentiated state of human ES cells by addition of bFGF/FGF-2 and the bone morphogenetic protein antagonist Noggin has also been reported (Xu et al., Nature Methods 2:185, 2005). Separately, it has been shown that simple addition of Glycogen Synthase Kinase (GSK)-3 inhibitor to culture medium can efficiently maintain the undifferentiated state of murine and human ES cells without addition of growth factors or the like and without using feeder cells (Sato et al., Nature Med. 10:55, 2004 (Non-Patent document 5)).
Thus, while new methods are being proposed for culturing of pluripotent stem cells without the use of feeder cells, actual implementation and industrial use of such cells will require even greater convenience of pluripotent stem cell growth effects and culturing methods.
One well known factor that maintains the undifferentiated state of murine ES/EG cells and increases their proliferation potency is the LIF mentioned above, and while the LIF-related IL-6 family of molecules falls under this category (Yoshida et al., Mech. Dev. 45:163, 1994; Koshimizu et al., Development 122:1235, 1996), very few other examples have been reported. Recently, serum-free medium containing added bFGF/FGF-2 or SCF has been reported to notably promote the proliferation potency of human ES cells (International Patent Publication No. WO03/020920 (Patent document 2)).
Given the active, i.e., proliferating, nature of ES cells in comparison to other cell types, few attempts have been made to actually investigate their proliferation potency; however, the needs of regenerative medicine will require increased proliferation of such cells.
One of the problems currently encountered in culturing pluripotent stem cells is that the cells generally form tight colonies and are therefore difficult to handle for passaging and the like. Undifferentiated ES/EG cells are usually found in a condition with the cells firmly adhering to each other, forming colonies, i.e. cell masses with indistinct boundaries between cells. For provision of ES/EG cells for passaging or differentiation-inducing experiments, it is therefore necessary to disperse the colonies in as short a period as possible by treatment with protease solutions of trypsin or the like. When this is done, however, dispersion of the ES/EG cell colonies into individual cells requires relatively high-concentration protease treatment and/or vigorous mechanical stirring, and such procedures significantly reduce the viability and adhesion ability of the ES/EG cells.
Moreover, since ES/EG cells undergo spontaneous differentiation during continuous culturing in a clustered condition, they must be dispersed to single cells during passaging and the passaging must be carried out before colonies grow to an excessive size. Murine ES cells, for example, generally require each passaging to be conducted for 2-3 days, and if the passaging is not conducted by a suitable method, cells that have deviated from their undifferentiated state may appear in the cluster, rendering the cells unsuitable for use. This cannot be overcome simply by adding a sufficient amount of a factor that maintains the undifferentiated state of ES/EG cells, such as the LIF mentioned above or GSK-3 inhibitors, and excessive colony growth and cells with a differentiated phenotype are induced. Therefore, a method of growing ES/EG cells without formation of colonies, i.e., with the cells individually dispersed, is expected to be highly useful for providing ES/EG cells for industrial use. However, no such attempts or successes can be found to date.
In recent years, totipotent cells that can be produced from skin or organ cells without destroying embryos, i.e., induced pluripotent stem cells (iPS cells), have been produced (Patent Document 3, Patent Document 4 and Patent Document 5).
iPS cells have been established in mice and human. Since iPS cells can be obtained without an ethical problem of embryo destruction, and human iPS cells that have been produced using cells from a patient to be treated can be used for differentiation into his/her tissue cells, iPS cells are, especially in the field of regenerative medicine, expected to be a graft material with no rejection. The properties of iPS cells are similar to those of ES cells, and there are problems similar to those of ES cells as described above.
The present inventors have previously seeded F9 cells, an embryonal carcinoma cell line known to normally proliferate by colony formation, on a culture plate coated with E-cadherin (Nagaoka et al., Biotechnol. Lett. 24:1857, 2002 (Non-patent document 6)) and have found that this prevents formation of cell colonies (International Symposium on Biomaterials and Drug Delivery Systems, 2002 Apr. 14-16, Taipei, Taiwan; 1st Meeting of the Japanese Society for Regenerative Medicine, 2002 Apr. 18-19, Kyoto, Japan). Specifically, F9 cells exhibited a dispersed cell morphology on a culturing plate having E-cadherin, which is a known cell adhesion molecule for F9 cells, immobilized on an untreated polystyrene culturing plate (hereinafter, “E-cad plate”).
F9 cells exhibit a phenotype somewhat similar to ES cells, expressing alkaline phosphatase (hereinafter, ALP) or SSEA-1 and Oct-3/4, which are known as specific ES/EG cell markers (Lehtonen et al., Int. J. Dev. Biol. 33:105, 1989, Alonso et al., Int. J. Dev. Biol. 35:389, 1991). However, F9 cells do not require feeder cells or LIF for maintenance of the undifferentiated state of the cells, and therefore are different in their mechanism of maintaining undifferentiation. Moreover, whereas ES cells have triploblast differentiating potential to all three germ layers, the differentiation of F9 cells is limited to endodermal cells, and they are unable to form chimeras. In other words, although F9 cells are used as an ES/EG cell model system in some experiments, they differ from ES/EG cells in many aspects involving the culturing method and culturing conditions.
Thus, it was not possible to predict, based on the scientific evidence, whether an E-cad plate can be used in ES cell culturing methods that require no feeder cells, whether ES cells cultured by such methods can be passaged while maintaining their undifferentiated state and pluripotency, and whether the proliferation potency of the ES cells can be increased. In fact, the proliferation potency of F9 cells cultured on an E-cad plate is roughly equivalent to that of F9 cells cultured on a conventional cell culturing plate, and no data had been obtained to suggest that the proliferation potency of ES cells could thereby be increased.
E-cadherin is known to be expressed by undifferentiated murine ES cells, and it is also known that intercellular adhesion is notably inhibited with ES cells that lack E-cadherin gene expression due to gene modification (Larue et al., Development 122:3185, 1996). However, it has not yet been attempted to use E-cadherin as an adhesion substrate in an ES/EG cell culturing method.
In addition to the efficient culturing methods described above, when pluripotent stem cells such as ES cells are to be used as a laboratory material or for production of regenerative medicine products, it is also necessary to design methods for efficiently introducing selected exogenous genes into the cells and expressing them. In particular, one strategy for applying ES cells in regenerative medicine for treatment of various diseases is to modify the cell properties, such as proliferation and differentiation potency or the drug sensitivity, and this can be satisfactorily realized by introducing and expressing appropriate exogenous genes in the cells. In the case of murine ES cells, it is widely known that genes can be artificially modified to produce transgenic mice or knockout mice, for which efficient gene transfer methods are especially useful.
Ordinary transfer of exogenous genes into cells is frequently accomplished using agents such as calcium phosphate, DEAE-dextran and cationic lipid preparations. However, application of such methods to ES cells is known to result in lower efficiency than for other cell types (Lakshmipathy et al., Stem Cells 22:531, 2004 (Non-patent document 8)). Methods using various viral vectors for transfer of exogenous genes have also been reported. For example, retroviral vectors (Chemy et al., Mol. Cell. Biol., 20:7419, 2000), adenovirus vectors (Smith-Arica et al., Cloning Stem Cells 5:51, 2003), lentivirus vectors (Amaguchi et al. J. Virol. 74:10778, 2000; Asano et al., Mol. Ther. 6:162, 2002; International Patent Publication No. WO02/101057), and Sendai virus vectors (Sasaki et al., Gene Ther. 12:203, 2005; Japanese Unexamined Patent Publication No. 2004-344001) are publicly known. Nevertheless, the construction and preparation of viral vectors require relatively complex and time consuming, while biological safety is also an issue, depending on the virus, and therefore such methods are neither convenient nor universally employed.
Consequently, exogenous gene transfer into ES cells is most commonly carried out by a method known as electroporation. This technique involves application of an electrical pulse to cells to transiently open pores in the cell membranes for introduction of an exogenous gene into the cells, and it is a highly flexible method. Recently, an improved technique called nucleofection has been established, whereby an exogenous gene is transferred directly into cell nuclei to achieve significantly higher expression efficiency (Lorenz et al., Biotech. Lett. 26:1589, 2004; Lakshmipathy et al., Stem Cells 22:531, 2004 (Non-patent document 8)). However, this method requires a special electrical pulse-generating device, and it is not easy to prepare the optimal conditions. Furthermore, it is necessary to first treat the cells with a protease such as trypsin to disperse the individual cells, and therefore the cell toxicity is relatively high. Thus, the most useful gene transfer methods for pluripotent stem cells such as ES cells would be methods using gene transfer agents that are inexpensive and convenient to prepare, and would allow efficient transfer of exogenous genes into cells being cultured in an incubator.
Embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) can become unlimited sources for differentiated cells including cells having a neural function, and are representative means promising for overcoming many human diseases. In embryonic development, neurons are generated from neuroectoderm progenitors. Efficient production of these ectodermal progenitor cells can allow on-demand production of various subtypes of neurons.
Many studies have been made in order to produce a specified lineage of neural cells from ES cells or iPS cells. Most of protocols for neural differentiation of ES cells are dependent on formation of so-called embryoid bodies (EBs) or cell clusters such as a spherical neural stem cell mass, at the beginning of differentiation. The studies on induction into a specified lineage of neural cells look promising at first, but subsequent studies proved that the neural cell populations obtained from ES cells or iPS cells contain not only various subtypes of neural cells but also non-neural cells including undifferentiated cells.